Alce TM, Popik W, Pitha PM; International Conference on AIDS.

Int Conf AIDS. 2002 Jul 7-12; 14: abstract no. MoPeA3031.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Univeristy, Baltimore, United States

The receptor complex essential for human immunodeficiency virus type I (HIV-1) attachment and entry is composed of CD4 and a chemokine coreceptor. T cell tropic HIV-1 uses CD4 and not CXCR4 to induce signal transduction pathways that augment viral replication. Binding of virus to surface CD4 rapidly induces association of the tyrosine kinase Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras independent manner. The importance of this interaction following virus binding is demonstrated by the requirement of functional CD4 for activation of MEK/ERK MAP kinase signaling and stimulation of the DNA binding activity of several transcription factors including AP-1, NF-kappaB and C/EBP. In addition, overexpression of a constitutively active form of Raf-1 in Jurkat T cells stimulates HIV-1 LTR promoter activity and significantly enhances viral replication. Consequently, we are testing the hypothesis that a peptide designed to act as a competitive inhibitor preventing Lck-Raf-1 association is likely to prevent HIV-1 induced aberrant expression of cytokine and chemokine genes and inhibit HIV replication. With the aim of developing a pseudosubstrate inhibitor, we have used GST pulldown assays to identify a region of Raf-1 (aa 149-331) that interacts in vitro with Lck. In addition, we have similarly shown that aa 67-116 in the SH3 domain of Lck interact with full length Raf-1. Mutations of key regulatory amino acids in Lck, including a proline to leucine mutation in the SH3 domain do not abrogate association with Raf-1 in vivo as determined by co-immunoprecipitation. HIV TAT-mediated protein transduction is the approach used to introduce these peptides into cells. The Lck-interacting fragment of Raf-1 has been expressed as a recombinant TAT-Raf fusion protein that is taken up by T cells. Here, we show the effect of this inhibitory peptide on HIV-1 replication, expression of inflammatory cytokine/chemokine genes, and abrogation of Lck-Raf-1 colocalization

Publication Types:

• Meeting Abstracts

Keywords:

• Antigens, CD4
• DNA Replication
• Gene Products, tat
• Genes, src
• HIV Long Terminal Repeat
• HIV-1
• Humans
• In Vitro
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
• NF-kappa B
• Protein-Tyrosine Kinases
• Proto-Oncogene Proteins c-raf
• Receptors, CXCR4
• Signal Transduction
• T-Lymphocytes
• Transcription Factor AP-1
• Transcription Factor RelA
• genetics
• immunology

Other ID:

• GWAIDS0016684

UI: 102254182

From Meeting Abstracts